The goal of this SBIR Phase I project is the development of a new drug discovery platform for screening new inhibitors of viral polymerases and for studying mechanisms of action of the prospective pharmaceutical compounds. The main innovation in this project is the proposed concept of a multi-enzyme assay, which is able to measure efficiency and identify the mechanism of action of a viral polymerase inhibitor in a single experiment using a small reaction volume in realtime detection mode using widely available commercial instruments (luminometers). The proposed platform is superior over the existing methods in that it substantially reduces the cost of reagents, reduces the assay time from days to minutes and is adaptable to high-throughput format for application in large and small scale drug discovery projects. The proposed SBIR project is in response to the NIH call for new approaches aimed at reducing costs and increasing speed of preclinical drug development. PUBLIC HEALTH RELEVANCE: Antiviral therapy represents an important frontier for development of potential lifesaving products. Worldwide, millions of people suffer from viral infections each year. Approximately 360 million people worldwide suffer from hepatitis B virus, the major cause of liver cancer. Worldwide more than 170 million people are infected with hepatitis C, the leading cause of acute liver inflammation and liver cancer. Around the world more than 42 million people are infected with lifethreatening human immunodeficiency virus (HIV), including more than 3 million children. To address the growing medical needs, there is a steady increase in the number of antiviral compounds in development. The proposed SBIR project will create a knowledge base and establish a new technology, which reduces the cost and time of developing pharmaceutical antiviral products with a large health impact in the USA and around the world.